定量检测 IL-2-HSA 融合蛋白双抗体夹心 ELISA 法的建立

目的:通过抗体配对方法,建立能高特异、高灵敏地定量检测食蟹猴体内 IL-2-HSA 融合蛋白浓度的双抗体夹心 ELISA 法.方法:以 IL-2单克隆抗体为包被抗体、IL-2-HSA 融合蛋白为夹心抗、生物素标记的 HSA 为检测抗体,一抗和二抗的工作浓度分别为8μg/mL 和1∶5000,HRP 标记的亲和素为1∶200.结果:IL-2-HSA 融合蛋白标准品的曲线范围为3.9~250 ng/mL,最低检测限为3.9 ng/mL,与 IL-2、HSA、GLP-1/HSA 和 CD20单抗均无交叉反应,方法的回收率为98.9%~101.5%,批内和批间准确度分别为96.1%~98.3%和93.9%~105.4%.结论:本方法符合新生物制品临床前药代动力学研究指导则的要求,可用 IL-2-HSA 融合蛋白在临床前药代动力学试验的定量检测.     

人胰高糖素样肽-1及其突变体在大肠杆菌BL21中的表达与生物活性研究

以人胰高糖素样肽-1（human glucagons-like peptide-1,GLP-1））为研究对象,以克隆载体pPblu2SKP/（GLP-1A2G）2基因为模板,利用PCR扩增获得GLP-1与其突变体GLP-1A2G的编码基因,并将其插入原核表达质粒pGEX-4T-1中,利用氯化钙法将其转入表达宿主大肠杆菌（Escherichia coli）BL21（DE3）中诱导表达,经IPTG诱导后,收集菌体。菌体经超声破碎后,裂解液用GST亲和层析纯化得到融合蛋白,经凝血酶酶解,用Superdex G-75凝胶层析,得到GLP-1及其突变体GLP-1A2G的样品,经SDS-PAGE电泳测定,样品纯度〉90%。小鼠糖耐量试验表明,相对于空白对照组而言,GLP-1及其突变体GLP-1A2G可以明显地控制血糖的水平,两者的生物活性并无明显差异。由于突变体GLP-1A2G较GLP-1可以更加有效的抵制DPPⅣ的降解,提示突变体GLP-1A2G较GLP-1在白蛋白融合或PEG修饰等方面具有更大的优势。     

人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达

目的：在巴斯德毕赤酵母中表达有降糖活性的人胰高血糖素样肽-1（hGLP-1）突变体（2Gly-hGLP-1）与人血清白蛋白（HSA）的融合蛋白。方法：为将GLP-1氨基酸序列第2位的丙氨酸（Ala）定点突变为甘氨酸（Gly）,根据毕赤酵母偏爱密码子合成编码2Gly-hGLP-1的基因;采用重叠PCR法拼接2Gly-hGLP-1和HSA的基因,使得2Gly-hGLP-1的C端与HSA的N端通过甘氨酸五肽接头连接;将该融合基因插入表达载体pPIC9构建为重组载体pPIC9/2Gly-hGLP-1-HSA,电击转化至毕赤酵母GS115细胞,通过表型筛选和诱导表达实验获得高效表达菌株;工程菌在5L发酵罐中培养后,对发酵产物进行分离纯化和生物学活性分析。结果：融合蛋白在5L发酵罐中的表达量约为200mg/L,经纯化后纯度可达95%以上;小鼠糖耐量实验表明该融合蛋白具有明显的控血糖活性。结论：在毕赤酵母中分泌表达的融合蛋白2Gly-hGLP-1-HSA具有降血糖活性。     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白（GLP-1/HSA）,表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4 h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

检测HSA—GLP-1融合蛋白双抗体夹心ELISA方法的建立

目的：为检测血清中HSA-GLP-1融合蛋白整体分子的浓度,建立一种特异、灵敏的定量检测食蟹猴体内HSA-GLP-1融合蛋白浓度的双抗体夹心ELISA的方法。方法：采用双抗体夹心ELISA方法,以GLP-1单克隆抗体为包被抗体、HSA-GLP-1融合蛋白为夹心抗原、anti-HSA-Biotin为检测抗体,用Streptavidin-HRP进行免疫放大,TMB显色。结果：建立了检测HSA-GLP-1融合蛋白的ELISA方法,其线性范围为15.6~1000 ng/mL,最低检测限为15.6 ng/mL,与GLP-1、HSA、IL2-HSA均无交叉反应,板内和板间精密度均小于15%,准确度为±15%,冻融稳定性和稀释稳定性良好。结论：建立的HSA-GLP-1蛋白检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于HSA-GLP-1融合蛋白在临床前药代动力学试验的定量检测。     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白(GLP-1/HSA),表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

利用GAP启动子在毕赤酵母中表达与纯化GLP-1类似物

胰高血糖素样肽-1(GLP-1)能提高II型糖尿病患者β胰腺细胞的胰岛素分泌量并能促进β胰腺细胞增殖,是潜在的糖尿病治疗药物。设计一种GLP-1类似物AGGH,即GLP-1(A2G)的二联体与人血清白蛋白(HSA)的N端连接,并在融合蛋白GGH前添加一个丙氨酸(A)。PCR获得融合基因aggh,将融合基因连接到p GAPZαA质粒中。在酵母中利用甘油醛三磷酸脱氢酶(GAP)启动子组成型表达外源蛋白AGGH。研究结果显示:筛选获得表达重组菌株,基因组PCR和western-blot验证正确;以葡萄糖为最优碳源培养下,表达量达到68 mg/L;5 L发酵罐中,发酵52 h蛋白产量最高达238 mg/L。蛋白经四步纯化后,获得纯度为95.8%的AGGH融合蛋白。与利用醇氧化酶1(AOX1)启动子表达的AGGH比较,发现两者产量和活性没有明显差异。但是,GAP启动子表达获得AGGH融合蛋白更加方便,且发酵时间减少了27.8%(20 h)。     

基因重组胰高血糖素样肽-1衍生多肽的表达和产物纯化与鉴定

为研究利用基因重组方法生产人胰高血糖素样肽-1（GLP-1）衍生多肽的最佳表达及纯化条件,选用大肠杆菌偏爱密码子,以含人GLP-1的质粒为模板,用PCR方法合成全长人GLP-1衍生多肽基因,并定向插入到高效表达载体pMFH中,用大肠杆菌BL21进行表达,融合蛋白经Ni-NTA柱纯化后,用C18 Sep-Pak 反相柱脱盐,然后融合蛋白经甲酸水解,水解产物经Ni-NTA柱和高效液相色谱（HPLC）纯化制备后,目的肽由质谱鉴定。 实验结果表明：利用载体pMFH在BL21中,GLP-1衍生物的最佳诱导表达温度为37℃、诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）的最佳浓度为0.6mmol/L,最佳诱导表达时间为6h;HPLC分析和制备GLP-1衍生物最佳条件为：流动相A(10% CNCH3∶90% H2O,0.1％TFA),流动相B(100% CNCH3,0.1% TFA),流速1ml／min,30 min线性梯度洗脱,B相至70%,检测波长280nm;质谱鉴定GLP-1衍生物的分子量为5.492kDa,与理论值相符合。在最佳表达及纯化条件下可得GLP-1衍生多肽的产量可达到11.6mg/L发酵产物,纯度≥98%。     

大肠杆菌表达Trx-rPA融合蛋白的复性纯化

大肠杆菌高密度发酵以包涵体形式表达融合蛋白Trx-rPA,表达量22%。包涵体蛋白洗涤后经金属螯合层析纯化,纯度达80%以上。经胱氨酸衍生,以脉冲加样形式复性,复性率可高达30%。经ETI-Sepharose纯化,复性的融合蛋白生物活性可达3.5×105IU/mgPr.。融合蛋白可被rEK酶切释放rPA,酶切效率达85%以上。酶切液经IDA-Sepharose和SP-Sepharose层析纯化,rPA纯度达98%以上,生物活性50万IU/mgPr.。1L发酵液经分离、复性及纯化后,可得高纯度rPA300mg以上。     

长效GLP-1-IgG2σ Fc融合蛋白的真核表达与生物活性鉴定

目的:在真核细胞中融合表达胰高血糖素样肽1(GLP-1),纯化后探究产物的生物学活性。方法:通过预留酶切位点将合成的GLP-1-Ig G2σFc融合蛋白表达序列连入表达载体质粒,用电转方法将线性化质粒稳定转染CHO-K1细胞,通过亚克隆筛选出蛋白高表达细胞株;收集高表达细胞株培养上清并初步纯化,采用口服葡萄糖耐量试验(OGTT)探究GLP-1融合蛋白降低血糖的生物学活性。结果:经过一轮亚克隆,筛选得到的细胞株的GLP-1融合蛋白产量达1 g/L,OGTT实验结果表明GLP-1融合蛋白具有降血糖生物活性。结论:GLP-1变体和Ig G2σ的Fc段连接是一种可行的融合蛋白形式,融合蛋白具有GLP-1受体激动剂的降糖作用,并且细胞株的筛选方式和初步纯化方法也具有可行性,为后续的体内、外活性研究和纯化方法确定提供了基础。     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.